CN116893227A - Method for constructing fingerprints of semen plantaginis, salinized semen plantaginis and stir-fried semen plantaginis and identification method thereof - Google Patents
Method for constructing fingerprints of semen plantaginis, salinized semen plantaginis and stir-fried semen plantaginis and identification method thereof Download PDFInfo
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- CN116893227A CN116893227A CN202310703454.4A CN202310703454A CN116893227A CN 116893227 A CN116893227 A CN 116893227A CN 202310703454 A CN202310703454 A CN 202310703454A CN 116893227 A CN116893227 A CN 116893227A
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- semen plantaginis
- plantain seed
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- 238000003672 processing method Methods 0.000 description 1
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- 239000012088 reference solution Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
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- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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- KDSWDGKIENPKLB-QJDQKFITSA-N verbascoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)CCC=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O KDSWDGKIENPKLB-QJDQKFITSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The application relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a fingerprint of plantain seed, salted plantain seed or fried plantain seed and an identification method thereof. The construction method comprises the following steps: providing a test solution: extracting the sample with an extraction solvent to obtain an extract; dispersing the extract in water, and extracting with ethyl acetate; taking a sample solution for chromatographic analysis, wherein the conditions comprise: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution with the volume concentration of 0.05-0.2%, and gradient elution is adopted. The fingerprints of the plantain seed, the salted plantain seed and the fried plantain seed prepared by the construction method have rich characteristic peak information, relate to various effective components with the consistency of the efficacy with the traditional efficacy of traditional Chinese medicinal materials, and can reflect the quality of the plantain seed and the processed products thereof as a basis for quality control.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of fingerprints of plantain seed, salted plantain seed and fried plantain seed and an identification method thereof.
Background
The traditional Chinese medicine plantain seed is a dry mature seed of plantain (Plantago asiatica L.) or plantain (Plantago depressa Willd.) of plantain family, is firstly carried in Shennong's herbal channels, has sweet taste and slightly cold nature, has the effects of inducing diuresis to treat stranguria, excreting dampness to arrest diarrhea, improving eyesight and eliminating phlegm, and is mainly used for treating heat stranguria pain, edema distention, summer-heat-dampness diarrhea, conjunctival congestion and swelling pain, phlegm-heat cough and the like. Modern processing methods of semen plantaginis mainly comprise cleaning, stir-frying and salt preparation, and raw materials are good at inducing diuresis for treating stranguria, cold property is weakened after processing, and salt semen plantaginis is good at excreting dampness and relieving diarrhea, and stir-fried semen plantaginis is good at eliminating phlegm and relieving cough. The basis of the pharmacodynamic action of the traditional Chinese medicine is the reasonable and organic combination of various active ingredients, and the mode is that the traditional Chinese medicine acts on multiple targets related to diseases in a body in multiple ways, and plays an integral regulation role on the body. Thus, the difference in the efficacy of psyllium and the processed product is understood to be the difference in the inherent chemical components.
The pharmacopoeia 2020 specifies the quality detection terms geniposidic acid and acteoside of plantago asiatica. Geniposide is iridoid glycoside compound, acteoside is called acteoside, and acteoside is phenethyl alcohol glycoside compound. The geniposide occupies higher duty ratio in the plantago seed, so the response value of the geniposide in the fingerprint is higher, other peaks are hidden or can not be clearly presented, and the chemical information reflected by the fingerprint is not comprehensive enough. The two compounds mainly show activities of resisting sugar, resisting oxidation, resisting inflammation and the like, and no related efficacy report on relieving cough, relieving asthma, eliminating phlegm and easing pain is seen. The traditional efficacy is a summary of the effectiveness of the traditional Chinese medicine and is also the basis of clinical medication, and it is necessary to develop quality markers which have the consistency with the traditional efficacy.
The chemical components of the plantain seed comprise polysaccharides, phenethyl alcohol glycosides, iridoids, flavonoids, alkaloids, triterpenes, sterols and other compounds, and modern pharmacological researches show that the phenethyl alcohol glycosides in the plantain seed have the functions of resisting oxidation, promoting urination, regulating immunity, resisting inflammation, resisting bacteria, protecting liver and the like, the flavonoid components have the functions of relieving cough, relieving asthma, eliminating phlegm, resisting inflammation and easing pain, and the compounds are consistent with the traditional efficacy of the plantain seed, are the main pharmacodynamic substance basis of the traditional efficacy of the plantain seed, and are also the main way and important basis for screening the quality markers of the plantain seed. The fingerprint spectrum of traditional Chinese medicine refers to the spectrum which is obtained by adopting a certain analysis method and can reflect the overall characteristics of traditional Chinese medicine after the traditional Chinese medicine is properly treated. According to the purpose of quality control, it can be classified into fingerprint and characteristic spectrum. The fingerprint is based on the overall information of the fingerprint and is used for overall evaluation of the quality of the traditional Chinese medicine; the characteristic map is to select some important characteristic information in the map as the identification means for controlling the quality of the traditional Chinese medicine. The fingerprint and characteristic spectrum technology of traditional Chinese medicine can integrally and macroscopically characterize the characteristics of main chemical components of a tested sample, and is one of the currently accepted means most suitable for quality control of internal substance groups of traditional Chinese medicine and natural medicine. The detection and identification of the plantain seed and the processed product thereof can be realized by establishing the fingerprint spectrum of the plantain seed and the processed product thereof.
Researches show that the proportion of organic acid in the whole body of the processed semen plantaginis is greatly increased, and meanwhile, components such as phenylethanoid glycosides, flavonoids and the like are easy to decompose or convert in the processing process, but the genipin Ping Gansuan peak in the fingerprint obtained by adopting a conventional extraction mode is very high, so that other components are difficult to present, and the identification of the semen plantaginis or processed products thereof is not facilitated. The traditional mode of taking semen plantaginis as a medicament is water decoction, and it is generally considered that the semen plantaginis decoction should be analyzed to study the spectral efficiency relationship. However, modern medical research shows that the ethyl acetate part of the plantain seed is a key drug effect part with the functions of promoting urination, diminishing inflammation and relieving cough, and research reports that the ethyl acetate extraction part has good antioxidant activity, which is consistent with the traditional efficacy of the plantain seed, and has important significance for quality control of the ethyl acetate part of the plantain seed.
In view of this, the technical scheme of the application is specifically proposed.
Disclosure of Invention
Based on the above, the application aims to provide a construction method of a characteristic map of plantain seed, salted plantain seed or fried plantain seed, and an identification method of plantain seed, salted plantain seed and fried plantain seed.
Specifically, the fingerprint construction method comprises the following steps:
Providing a sample solution comprising the steps of: extracting the sample with an extraction solvent to obtain an extract; dispersing the extract in water, and adding ethyl acetate for extraction; the sample is semen plantaginis, salted semen plantaginis or parched semen plantaginis;
taking the sample solution for liquid chromatography, wherein the conditions of the liquid chromatography comprise:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is employed, the procedure of which comprises:
the volume percentage of the mobile phase A is increased from 10% to 16% after 0min to 5min,
5-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15-20 min, wherein the volume percentage of the mobile phase A is increased from 18% to 25%,
20-25 min, wherein the volume percentage of the mobile phase A is increased from 25% to 45%,
25-30 min, wherein the volume percentage of the mobile phase A is increased from 45% to 60%,
30-40 min, wherein the volume percentage of the mobile phase A is increased from 60% to 70%,
the volume percentage of the mobile phase A is reduced from 70% to 10% in 40-41 min,
41-50 min, wherein the volume percentage of the mobile phase A is maintained to be 10%.
The identification method comprises the following steps:
taking the standard substances of semen plantaginis, salted semen plantaginis and stir-fried semen plantaginis as reference substances, and respectively preparing corresponding fingerprint patterns according to the construction method;
providing a solution to be tested, comprising the steps of: extracting the to-be-detected product with an extraction solvent to obtain an extract; dispersing the extract in water, and adding ethyl acetate for extraction;
performing liquid chromatography analysis on the solution to be detected to obtain a chromatographic spectrum of the to-be-detected product, and respectively comparing the chromatographic spectrum of the to-be-detected product with the fingerprint spectrum of the plantain seed reference product, the fingerprint spectrum of the salted plantain seed reference product and the fingerprint spectrum of the fried plantain seed reference product;
the conditions of the liquid chromatography include:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is employed, the procedure of which comprises:
the volume percentage of the mobile phase A is increased from 10% to 16% after 0min to 5min,
5-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15-20 min, wherein the volume percentage of the mobile phase A is increased from 18% to 25%,
20-25 min, wherein the volume percentage of the mobile phase A is increased from 25% to 45%,
25-30 min, wherein the volume percentage of the mobile phase A is increased from 45% to 60%,
30-40 min, wherein the volume percentage of the mobile phase A is increased from 60% to 70%,
the volume percentage of the mobile phase A is reduced from 70% to 10% in 40-41 min,
41-50 min, wherein the volume percentage of the mobile phase A is maintained to be 10%.
The fingerprint of semen plantaginis, salted semen plantaginis and stir-fried semen plantaginis prepared by the construction method has rich characteristic peak information, relates to various effective components with efficacy consistency with traditional Chinese medicinal materials, comprises various categories such as flavonoids, iridoids, phenylethanoid glycosides and the like, can reflect the quality of semen plantaginis and processed products thereof on the whole, is taken as a basis for quality control, is beneficial to establishing a spectrum effect relation with practical significance, and provides theoretical basis for clinical medicine.
According to the application, the fingerprints of the semen plantaginis, the salted semen plantaginis and the fried semen plantaginis are prepared by establishing proper chromatographic conditions, and the characteristic indexes of the semen plantaginis, the salted semen plantaginis and the fried semen plantaginis are respectively obtained, and have obvious differences, so that the identification can be well realized, wherein the identification components in the fingerprints of the semen plantaginis comprise geniposide, ferulic acid, plantain, luteolin, verbascoside, plantain D, fissistigma, apigenin-7-O-glucuronide and apigenin; the finger print of semen plantaginis comprises geniposide, protocatechuic acid, ferulic acid, acteoside, caulis Akebiae phenethyl alcohol glycoside B, plantain glycoside D, iso-acteoside, fisetin, apigenin-7-O-glucuronide, luteolin and apigenin; the finger print of semen plantaginis comprises geniposide, protocatechuic acid, ferulic acid, acteoside, caulis Akebiae phenethyl alcohol glycoside B, plantain glycoside D, iso-acteoside, fisetin, apigenin-7-O-glucuronide, luteolin and apigenin. The fingerprint can also be combined with statistical analysis, chemical pattern recognition research, colorimetric value research and antioxidant activity measurement to realize comprehensive distinction of semen plantaginis and its processed products of salted semen plantaginis and parched semen plantaginis from the aspects of internal components, appearance color and pharmacodynamic activity.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a superimposed graph of the UPLC spectra of 12 batches of psyllium from example 1;
FIG. 2 is a superimposed graph of UPLC spectra of 12 batches of parched semen plantaginis of example 1;
FIG. 3 is a superimposed graph of the UPLC spectra of 12 batches of psyllium in example 1;
FIG. 4 shows the results of comparison of the fingerprints of semen plantaginis, semen plantaginis salt, semen plantaginis preparata and the chromatograms of the control in example 1, wherein A is the chromatogram of the mixed control, B is the fingerprint of semen plantaginis, C is the fingerprint of semen plantaginis preparata, and D is the fingerprint of semen plantaginis salt;
FIG. 5 is a graph showing the principal component analysis of the UPLC detection results of 12 batches of semen plantaginis, salted semen plantaginis and parched semen plantaginis in example 1;
FIG. 6 is a graph showing the results of a cluster analysis of UPLC detection results of 12 batches of semen plantaginis, salted semen plantaginis and stir-fried semen plantaginis in example 1;
FIG. 7 is a graph showing the results of the orthorhombic least squares discriminant analysis of UPLC detection results of 12 batches of psyllium seeds, salted psyllium seeds and fried psyllium seeds in example 1;
FIG. 8 is a graph showing the results of a variable importance projection analysis of UPLC detection results of 12 batches of psyllium, salted psyllium and fried psyllium in example 1;
FIG. 9 is a normalized regression coefficient of partial least squares regression analysis of UPLC detection results of 12 batches of semen plantaginis, parched semen plantaginis and salted semen plantaginis in example 1;
FIG. 10 is a graph showing the comparison of fingerprints of semen plantaginis, salted semen plantaginis, and poor semen plantaginis prepared under different test solution preparation conditions in comparative example 1;
FIG. 11 is a graph showing the comparison of fingerprints of semen plantaginis obtained under different mobile phase elution conditions in comparative example 2, wherein 11A corresponds to elution procedures 1-3 and 11B corresponds to elution procedures 4-6.
Detailed Description
The application is further illustrated below in conjunction with the embodiments, examples and figures. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it is to be understood that various changes and modifications may be made by one skilled in the art after reading the teachings of the application, and such equivalents are intended to fall within the scope of the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Terminology
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other.
Herein, "preferred", "better", etc. are merely embodiments or examples that describe better results, and it should be understood that they do not limit the scope of the application.
In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.
In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. When a numerical range merely points to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, unless expressly stated otherwise. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the present application, the weight may be a mass unit known in the chemical industry field such as mu g, mg, g, kg.
In one aspect of the application, a method for constructing the fingerprints of semen plantaginis, salted semen plantaginis and stir-fried semen plantaginis is provided, the fingerprints prepared by the method have systematicness, characteristics and stability, can more comprehensively reflect the internal chemical information of semen plantaginis medicinal materials or processed products thereof, and the characteristic information reflected by the fingerprints has correlation with the traditional efficacy of traditional Chinese medicines, can provide clinical medicinal references, and is simple and easy to operate, short in time consumption and cost-saving.
In the detection and/or identification methods of semen plantaginis or processed products thereof disclosed at present, the problem that characteristic peaks of other components are difficult to identify due to the fact that the response value of geniposide as an index component is too high is commonly existed. This problem is difficult to avoid because of the higher content of geniposide in psyllium. In addition, the chemical components of the plantain seed before and after processing have been changed, and researches show that organic acids in the salted or fried plantain seed are greatly increased, and the components such as phenylethanoid glycosides, flavonoids and the like are decomposed or converted, so that the changes of the components are difficult to be simultaneously reflected in the fingerprint.
In order to overcome the problems, the method adopts the ethyl acetate extraction mode to prepare the sample solution, so that the preservation of geniposide acid is reduced while the medium and small polar components are enriched, and the information of the fingerprint of the plantain seed can be more enriched.
In some embodiments, the method of constructing psyllium, salted psyllium, or roasted psyllium comprises the steps of:
providing a sample solution comprising the steps of: extracting the sample with an extraction solvent to obtain an extract; dispersing the extract in water, and extracting with ethyl acetate; the sample is semen plantaginis, salted semen plantaginis or parched semen plantaginis;
Taking a sample solution for liquid chromatography, wherein the conditions of the liquid chromatography include:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is employed, the procedure of which comprises:
0-5 min, the volume percentage of the mobile phase A is increased from 10% to 16%,
5-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15-20 min, the volume percentage of the mobile phase A is increased from 18% to 25%,
20 min-25 min, the volume percentage of the mobile phase A is increased from 25% to 45%,
25 min-30 min, the volume percentage of the mobile phase A is increased from 45% to 60%,
30-40 min, the volume percentage of the mobile phase A is increased from 60% to 70%,
the volume percentage of the mobile phase A is reduced from 70 percent to 10 percent in 40 to 41 minutes,
41-50 min, and the volume percentage of the mobile phase A is maintained to be 10%.
Preferably, the liquid chromatography is performed using an ultra high performance liquid chromatography analyzer (UPLC).
In some embodiments, the extraction solvent comprises methanol and the extraction means comprises ultrasound.
In some embodiments, the conditions of ultrasound include at least one of:
(1) The power of the ultrasonic wave is 200W-300W,
(2) The frequency of the ultrasonic wave is 30 kHz-50 kHz, and
(3) The ultrasonic time is 50 min-70 min.
In some embodiments, the conditions under which the test solution is provided include at least one of:
(1) The volume ratio of water to ethyl acetate is 1: (1-3),
(2) The mass volume ratio of the sample and the extracting solvent is 1 g/(20-30) mL, and
(3) The mass volume ratio of the test sample and the ethyl acetate is 1 g/(20-60) mL.
In some embodiments, in the chromatographic analysis:
0 min-10.5 min, the detection wavelength is 240 nm-250 nm,
10.5 min-35.5 min, the detection wavelength is 320 nm-340 nm,
35.5-50 min, and the detection wavelength is 240-250 nm.
In some embodiments, the chromatographic conditions further comprise at least one of:
(1) The chromatographic column is a C18 chromatographic column,
(2) The chromatographic column has a length of 100 mm-150 mm, an inner diameter of 2 mm-2.2 mm and a particle size of 1.6 μm-1.9 μm,
(3) The flow rate is 0.28 mL/min-0.32 mL/min,
(4) Column temperature is 28-32 ℃, and
(5) The sample injection amount is 1-3 mu L.
In some embodiments, the chromatographic analysis uses a suitable control, which may be selected from geniposide, protocatechuic acid, ferulic acid, plantain, luteolin, acteoside, akebia stem phenethyl alcohol glycoside B, plantain D, iso-acteoside, rhoifolin, apigenin-7-O-glucuronide, luteolin, apigenin, or a combination of several.
Optionally, the sample is semen plantaginis, and the reference substance comprises at least one of geniposide, ferulic acid, plantain, luteolin, acteoside, plantain D, rhoifolin, apigenin-7-O-glucuronide and apigenin.
Optionally, the sample is semen plantaginis, and the reference substance comprises at least one of geniposide, protocatechuic acid, ferulic acid, acteoside, akebia phenylethanoid glycoside B, plantain glycoside D, iso-acteoside, stauroside, apigenin-7-O-glucuronide, luteolin and apigenin.
Optionally, the sample is semen plantaginis preparata, and the reference substance comprises at least one of geniposide, protocatechuic acid, ferulic acid, acteoside, akebia stem phenethyl alcohol glycoside B, plantain glycoside D, iso-acteoside, stauroside, apigenin-7-O-glucuronide, luteolin and apigenin.
In some embodiments, the reference is dissolved in a solvent to produce a reference solution, which is then chromatographed. Further, the solvent may be selected from methanol.
In some embodiments, the mass concentration of the reference in the solvent is from 30 μg/mL to 160 μg/mL.
In a further aspect of the present application, a method for identifying psyllium, salted psyllium and fried psyllium is provided, wherein the method comprises preparing fingerprints of psyllium, salted psyllium and fried psyllium preparations by establishing suitable chromatographic conditions, obtaining characteristic indexes of psyllium, salted psyllium and fried psyllium, and combining statistical analysis, chemical pattern recognition study, colorimetric value study and measurement of antioxidant activity, thereby realizing comprehensive differentiation of psyllium and its preparation, salted psyllium and fried psyllium from the aspects of internal composition, apparent color and pharmacodynamic activity.
In some embodiments, the method of identifying psyllium, salted psyllium, and roasted psyllium comprises the steps of:
taking the standard substances of the plantain seed, the salted plantain seed and the fried plantain seed as reference substances, and respectively preparing corresponding fingerprint patterns according to the construction method of any technical scheme;
providing a solution to be tested, comprising the steps of: extracting the to-be-detected product with an extraction solvent to obtain an extract; dispersing the extract in water, and extracting with ethyl acetate;
taking the solution to be detected for liquid chromatography analysis to obtain a chromatographic spectrum of the to-be-detected product, and respectively comparing the chromatographic spectrum of the to-be-detected product with the fingerprint spectrum of the plantain seed reference substance, the fingerprint spectrum of the salted plantain seed reference substance and the fingerprint spectrum of the fried plantain seed reference substance;
the conditions for liquid chromatography include:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is used, and the gradient elution procedure comprises:
0 mm-5 min, the volume percentage of the mobile phase A is increased from 10% to 16%,
5 mm-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15 mm-20 min, the volume percentage of the mobile phase A is increased from 18% to 25%,
20 mm-25 min, the volume percentage of the mobile phase A is increased from 25% to 45%,
25 mm-30 min, the volume percentage of the mobile phase A is increased from 45% to 60%,
30 mm-40 min, the volume percentage of the mobile phase A is increased from 60% to 70%,
the volume percentage of the mobile phase A is reduced from 70 percent to 10 percent in 40 mm-41 min,
41 mm-50 min, the volume percentage of the mobile phase A is maintained to be 10%.
Preferably, the liquid chromatography is performed using an ultra high performance liquid chromatography analyzer (UPLC).
In some embodiments, the extraction solvent comprises methanol and the means of extraction comprises ultrasound.
In some embodiments, the conditions of ultrasound include at least one of:
(1) The power of the ultrasonic wave is 200W-300W,
(2) The frequency of the ultrasonic wave is 30 kHz-50 kHz, and
(3) The ultrasonic time is 50-70 min.
In some embodiments, the conditions under which the solution to be assayed is provided include at least one of:
(1) The volume ratio of water to ethyl acetate is 1: (1-3),
(2) The mass volume ratio of the sample and the extracting solvent is 1 g/(20-30) mL, and
(3) The mass volume ratio of the test sample and the ethyl acetate is 1 g/(20-60) mL.
In some embodiments, in the chromatographic analysis:
0 min-10.5 min, the detection wavelength is 240 nm-250 nm,
10.5 min-35.5 min, the detection wavelength is 320 nm-340 nm,
35.5-50 min, and the detection wavelength is 240-250 nm.
In some embodiments, the chromatographic conditions further comprise at least one of:
(1) The chromatographic column is a C18 chromatographic column,
(2) The chromatographic column has a length of 100 mm-150 mm, an inner diameter of 2 mm-2.2 mm and a particle size of 1.6 μm-1.9 μm,
(3) The flow rate is 0.28 mL/min-0.32 mL/min,
(4) Column temperature is 28-32 ℃, and
(5) The sample injection amount is 1-3 mu L.
In some embodiments, the identification method further comprises identifying a colorimetric value for the test agent.
In some embodiments, the colorimetric value identification comprises the steps of: respectively measuring the chromaticity values of the plantain seed, the salted plantain seed, the stir-fried plantain seed and the to-be-measured product by adopting a spectrocolorimeter;
examples of the measurement method include: the sample powder is evenly spread on a glass slide, pressed to a thickness of about 1mm, white is taken as a background, the aperture is measured to be 8mm, the visual angle is selected to be 2 degrees, after the white board is corrected, the color image of the decoction piece powder is collected, the chromaticity value is measured, and the average value is obtained after three times of parallel.
In some embodiments, the identification method further comprises performing an antioxidant activity assay on the test substance, and further performing identification based on the difference in antioxidant activity.
In some embodiments, the method of determining antioxidant activity comprises: the concentration of the sample solution was set to 60. Mu.g/mL based on the preliminary test, and the measurement was performed according to the method prescribed by the Grignard kit.
The operation steps of the measurement are as follows:
(1) Preparation of a reagent solution:
reagent one: the powder was dropped to the bottom by throwing several times before use, and each was dissolved by adding 0.49mL of distilled water.
And (2) a reagent II: before use, the powder falls into the bottom by throwing a few times, and 2.86mL of distilled water is added into each powder to be fully dissolved for standby.
Preparing an ABTS working solution: according to the sample size, the reagent I: the reagents II=1:1 are mixed in proportion, and after light-shielding reaction is carried out for 12 hours (after the reaction is finished within two days), the mixture is diluted by 20 to 30 times by absolute ethyl alcohol for standby, and each reagent and the formula thereof are shown in the table 1.
TABTS reaction System
Mixing the above solutions, standing in dark for 6min, transferring into a glass cuvette (optical path 1 cm), and reading absorbance at 734nm, respectively designated as A Blank space 、A Control And A Measurement And ABTS radical scavenging was calculated. ABTS radical scavenging% = [1- (a) Measurement -A Control )/A Blank space ]×100％。
In some embodiments, the step of constructing the fingerprint further comprises performing a multivariate statistical analysis of peak areas of the common peaks of the fingerprint.
In some embodiments, fingerprints of semen plantaginis, salted semen plantaginis and fried semen plantaginis are taken, the common peak area of each fingerprint is obtained, the moisture is reduced, SPSS25.0 software is respectively introduced for single-factor variance analysis, SIMCA14.l software is adopted for cluster analysis, principal component analysis and orthorhombic partial least squares discriminant analysis, and the diversity of the fingerprints of semen plantaginis, salted semen plantaginis and fried semen plantaginis is subjected to multivariate statistical analysis.
In some embodiments, the chromaticity values L, a, b of the plantain seed medicinal material and its processed decoction piece powder are measured by a spectrocolorimeter, and the total chromaticity value e= (L) is calculated according to the formula 2 +a* 2 +b* 2 ) 1/2 、ΔL*＝L*-L 0 *、Δa*＝a*-a 0 *、Δb*＝b*-b 0 * Color difference Δe= (Δl) 2 +Δa* 2 +Δb* 2 ) 1/2 Calculating E, deltaL, deltaa, deltab and DeltaE values of the respective samples, wherein the values are used for expressing the color change condition from the raw product to the processed product, and when DeltaE is 6-12, the color difference can be identified by naked eyes, and when DeltaE is 6-12>At 12, the color difference is significant.
In some embodiments, the determination of antioxidant activity comprises: diluting the test solution, determining the antioxidant activity of semen plantaginis medicinal material and its processed decoction pieces according to the prescription of the Grignard kit, and according to the formula: ABTS radical scavenging% = [1- (a) Measurement -A Control )/A Blank space ]The antioxidant capacity of the samples was calculated by x 100%.
In some embodiments, the spectral-efficiency relationship analysis includes: (1) The SIMCA14.1 software is utilized, the ABTS clearance of the semen plantaginis medicinal material and the processed decoction pieces thereof is taken as a dependent variable, the peak area of the common peak is taken as an independent variable after water is subtracted by conversion, the correlation analysis is carried out by adopting partial least squares regression analysis (PLSR), a regression equation is established, the regression coefficient is greater than 0, the positive correlation between the semen plantaginis medicinal material and the processed decoction pieces thereof is shown, the regression coefficient is less than 0, and the negative correlation between the semen plantaginis medicinal material and the antioxidant activity is shown; (2) Calculating the association degree by using the data averaged by the antioxidant activity as a parent sequence and the data averaged by the peak area of the common peak as a subsequence by using the spssau23.0 software, and indicating that the association degree between the parent sequence and the subsequence is larger when the association degree is larger than 0.8; when the association degree is between 0.6 and 0.8, the association degree is common; when the association degree is smaller than 0.6, the association degree of the two is smaller.
The following are some specific examples.
The experimental parameters not specified in the following specific examples are preferentially referred to the guidelines given in the present document, and may also be referred to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.
The starting materials and reagents referred to in the following specific examples may be obtained commercially or may be prepared by known means by those skilled in the art.
Example 1
1. Instrument and materials
1.1. Instrument for measuring and controlling the intensity of light
Waters ACQUITY ultra high performance liquid chromatography system (Wotech Co., U.S.); thermo Q-actual Focus liquid chromatography (Siemens, USA); analytical balance type ME204E, XP26 (Metrehler-Toli, switzerland); KQ-700DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); HWS28 electric heating constant temperature water bath (Shanghai-Heng technology Co., ltd.); 111B type two-by-two high speed traditional Chinese medicine pulverizer (Zhejiang Rui An Shi Yongli pharmaceutical machinery Co., ltd.); H22-X3 electric ceramic furnace (Jiuyang household appliances Co., ltd.); TS7700 type spectrocolorimeter (Sanhen, inc. of Sanhen, inc.); UV-2600 type ultraviolet spectrophotometer (Shimadzu corporation).
1.2. Material
Geniposide acid (lot number 11828-201805, purity 98.1%, national food and drug verification institute); ferulic acid (lot number: 110773-201915, purity 99.4%, national food and drug verification institute); plantain (lot No. 111914-202105, purity 96.0%, national food and drug institute); luteolin (lot number: 111720-202111, purity 96.6%, national institute of food and drug testing); acteoside (lot number: 111530-201914, purity 95.2%, national food and drug verification institute); acteoside (lot number: FF17B039, purity 98%, chengdu Poisson Biotechnology Co., ltd.); plantaginide D (lot number CFS202101, purity 98.0%, chemFaces); apigenin-7-O-glucuronide (lot number: 21062901, purity 98.29%, chenopodie Biotechnology Co., ltd.); luteolin (lot number: 111520-202006, purity 94.4%, national institute of food and drug testing); apigenin (lot number: 111901-202004, purity 99.4%, national food and drug verification institute); protocatechuic acid (lot number: 110809-201606, purity 97.7%, national food and drug verification institute); rhus verniciflua Stokes glycoside (lot number: 111919-201614, purity 95.5%, national food and drug verification institute); akebia phenylethanoid glycoside B (lot number: 111910-201604, purity 98.2%, national food and drug substance verification institute); chromatographic grade methanol, acetonitrile (merck, germany); chromatographic grade phosphoric acid (Tianjin, denou chemical Co., ltd.); the water was ultrapure water (from the laboratory Milli-Q ultrapure water system) and the remaining reagents were all analytically pure; ABTS free radical scavenging ability assay kit (cat# G0127F, soviet biosciences Inc.).
The 12 batches of psyllium were identified by the main pharmacist Sun Dongmei of the Guangdong party pharmaceutical Co., ltd as dry mature seeds of psyllium (Plantago asiatica L.) of the Plantaginaceae family, and the 12 batches of roasted and 12 batches of salted psyllium were self-made by the laboratory of the Guangdong party pharmaceutical Co. Specific sample information is shown in Table 2.
TABLE 2 sample Source information Table
2. Method and results
2.1. Preparation of fried plantain seed and salted plantain seed
2.1.1. Fried plantain seed
Taking semen plantaginis, parching under the first book of processing standards of decoction pieces of Chinese medicinal materials in Guangdong province until the color is slightly exploded and fragrance overflows, and cooling.
2.1.2. Salted semen plantaginis
Taking semen plantaginis, parching according to salt water roasting method (general rule 0213) under the condition of the first processed semen plantaginis of 2020 edition of Chinese pharmacopoeia 2020, spraying salt water, parching to dryness, taking out, and cooling.
2.2. Establishment of finger print of semen plantaginis and different processed products thereof
2.2.1. Chromatographic conditions
A Waters ACQUITY UPLC HSS T Column (2.1 mm. Times.150 mm,1.8 μm) was used; the mobile phase is acetonitrile (A) which is 0.1 percent phosphoric acid aqueous solution (B), gradient elution (0 to 5min,10 percent A, 5 to 15min,16 to 18 percent A, 15 to 20min,18 to 25 percent A, 20 to 25min,25 to 45 percent A, 25 to 30min,45 to 60 percent A, 30 to 40min,60 to 70 percent A, 40 to 41min,70 to 10 percent A, 41 to 50min,10 percent A); program detection wavelength: 245nm (0-10.5 min), 330nm (10.5-35.5 min), 245nm (35.5-50 min); the flow rate is 0.3mL/min; column temperature is 30 ℃; the sample injection amount was 2. Mu.L.
2.2.2. Preparation of control solution
Precisely weighing geniposide, protocatechuic acid, ferulic acid, plantain, luteolin, acteoside, akebia phenethyl alcohol glycoside B, plantain glycoside D, iso-acteoside, rhoifolin-7-O-glucuronide, luteolin and apigenin reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solutions with mass concentrations of 53.0721, 84.2174, 86.1798, 40.5120, 42.1176, 158.8888, 69.0346, 44.5900, 57.2320, 49.1825, 48.5553, 38.0432 and 62.6220 mug/mL respectively.
2.2.3. Preparation of test solutions
Taking about 1.0g of decoction piece powder (sieving with a second sieve), precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of methanol, performing ultrasonic treatment (250W, 40 kHz) for 60min, centrifuging, filtering, evaporating filtrate to dryness, adding 20mL of water into residues to dissolve, extracting with ethyl acetate for 3 times, 20mL each time, combining ethyl acetate, evaporating to dryness, dissolving residues with methanol, metering to 10mL, filtering, and taking subsequent filtrate.
2.2.4. Precision test
Taking semen plantaginis sample solution, continuously sampling and measuring for 6 times according to the chromatographic condition under the item of 2.2.1, taking 17 # acteoside chromatographic peak as reference peak (S), calculating the relative retention time RSD range of each common peak and S peak to be 0.06% -0.96%, and the relative peak area RSD range to be 0.09% -2.89%, which indicates that the instrument precision is good.
2.2.5. Stability test
Taking semen plantaginis sample solution, and respectively carrying out sample injection measurement after preparation for 0,2,4,8, 16 and 24 hours according to chromatographic conditions under the item of 2.2.1, and calculating the relative retention time RSD range of each common peak and S peak to be 0.08-1.20% and the relative peak area RSD range to be 0.31-2.53% by taking the 17 # acteoside chromatographic peak as a reference peak (S), so that the sample solution has good stability in 24 hours;
2.2.6. repeatability test
Taking a front sub-sample, preparing a test solution according to the method under the item "2.2.3", preparing 6 parts in parallel, carrying out sample injection measurement according to the chromatographic condition under the item "2.2.1", taking a 17 # acteoside chromatographic peak as a reference peak (S), and calculating the relative retention time RSD range of each common peak and the S peak to be 0.05-1.07% and the relative peak area RSD range to be 0.13-2.52%, thus showing that the method has good repeatability.
2.2.7. Fingerprint establishment and chromatographic peak identification
Taking 12 batches of semen plantaginis, parched semen plantaginis and salted semen plantaginis samples, preparing a sample solution according to the method under the item "2.2.3", sampling and detecting according to the chromatographic condition under the item "2.2.1", and collecting a chromatogram. Data processing is carried out by adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition, fingerprint is established, S01, C01 and Y01 fingerprints are respectively used as reference fingerprints, a median method is used as a reference fingerprint generation mode, and full-peak matching is carried out to respectively generate 12 batches of reference fingerprints of plantain seed, fried plantain seed and salicornia seed. The total of 12 batches of semen plantaginis is marked with 19 common peaks, and the total of 12 batches of stir-fried semen plantaginis and salted semen plantaginis is marked with 27 common peaks, and the results are shown in figures 1-3. Through mass spectrum accurate molecular weight and fragment ion comparison analysis, comparison with a reference substance retention time and an ultraviolet absorption spectrum shows that 13 main components are respectively peak 4 (geniposide), peak 5 (protocatechuic acid), peak 13 (ferulic acid), peak 14 (plantain), peak 16 (luteolin), peak 17 (acteoside), peak 18 (akebia phenethyl alcohol glycoside B), peak 19 (plantain D), peak 21 (acteoside), peak 22 (stauroside), peak 23 (apigenin-7-O-glucuronide), peak 28 (luteolin) and peak 29 (apigenin), and the result is shown in figure 4.
2.2.8. Similarity evaluation
The similarity of the fingerprints of 12 batches of plantain seed, fried plantain seed and salted plantain seed is calculated by taking the reference fingerprints of plantain seed, fried plantain seed and salted plantain seed respectively, and the results are shown in Table 3. The results show that the similarity of the semen plantaginis of 12 batches, the fried semen plantaginis and the salted semen plantaginis is more than 0.9, which shows that the difference among the semen plantaginis samples of different batches is smaller, and the quality among the processed samples is also more stable.
TABLE 3 sample similarity evaluation results
2.3. Fingerprint spectrum multivariate statistical analysis of semen plantaginis and different processed products thereof
2.3.1. Single factor analysis of variance
The peak areas of the psyllium and its various preparations (the peak areas of the partial deletion chromatograph were calculated as 0) were each introduced into SPSS25.0 for single-factor analysis of variance, and the results are shown in Table 4. The results show that the chromatographic peak changes of the semen plantaginis before and after processing are obvious. Wherein, compared with the crude product, 13 chromatographic peaks are added in the fingerprint of the processed semen plantaginis, namely, peaks 1-3, 6-12, 18, 20 and 28, and 4 peaks are deleted, namely, peak 14, 16, 23 and 31. In addition, except for the peak 25, the difference between the peak area of each chromatographic peak of the processed semen plantaginis and the raw semen plantaginis has statistical significance (P is less than 0.05); peak area differences of the fried plantain seed and the salted plantain seed of peak 6, peak 8, peak 12, peak 15, peak 17 (acteoside), peak 19 (acteoside D), peak 21 (acteoside), peak 22 (acteoside), peak 24, peak 26, peak 27 and peak 33 are all statistically significant (P < 0.05).
Table 4 analysis of variance of fingerprint peak area single factor (unit: mAU. Min)
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In table 4, compared with the psyllium, a p is less than 0.05; compared with the stir-fried plantain seed, b P＜0.05。
2.3.2. principal Component Analysis (PCA)
The peak areas of 12 batches of semen plantaginis, parched semen plantaginis and salted semen plantaginis (the peak areas of partial deletion chromatograph peak are calculated as 0) were introduced into SIMCA14.1, and the principal component analysis was performed. With the feature value >1 as a standard, 3 main components are extracted, the cumulative variance contribution rate is 83.5%, the prediction of the prompt model is good, and the information of the whole of 12 batches of plantain seeds, stir-fried plantain seeds and salted plantain seeds can be reflected, and the contribution rate of each main component is shown in table 5. The front 3 main components are used for establishing a coordinate system, the obtained PCA score diagrams of the plantain seed, the stir-fried plantain seed and the salted plantain seed are shown in fig. 5, and the diagram shows that the raw plantain seed and the processed product can be obviously divided into 2 groups, but partial samples of the stir-fried plantain seed and the salted plantain seed are overlapped with each other and cannot be well distinguished.
TABLE 5 principal component eigenvalues and variance contribution ratios
2.3.3. Cluster Analysis (HCA)
The peak areas of 12 batches of plantain seeds, fried plantain seeds and salted plantain seeds (the peak areas of partial deletion chromatograph are calculated as 0) are introduced into SIMCA14.1, and clustering analysis is carried out by adopting an inter-group connection method and taking square Euclidean distance as a classification basis. As shown in FIG. 6, when the clustering distance is 100, the semen plantaginis products and the processed products can be obviously classified into 2 types, but the fried semen plantaginis and the salted semen plantaginis cannot be clustered well, wherein Y06, Y11, Y04 and C01, C04 are clustered together, and Y09 and the rest batches of fried semen plantaginis are clustered together, which indicates that the similarity of the fried semen plantaginis and the salted semen plantaginis products is higher and basically consistent with the analysis result of the main component.
2.3.4. Orthogonal partial least squares discriminant analysis (OPLS-DA)
In order to screen the main marker components of the difference in the components of psyllium seed, fried psyllium seed and salted psyllium seed, 12 batches of peak areas of psyllium seed, fried psyllium seed and salted psyllium seed (the peak areas of partial deletion chromatograph peaks are calculated as 0) were introduced into SIMCA14.1 and subjected to orthorhombic partial least squares discriminant analysis. The variable projection importance (Variable Importance for the Projection, VIP value) of each index is arranged in size, and the index with VIP value >1 is selected as the main differential component for distinguishing plantain seed, fried plantain seed and salted plantain seed.
In the established OPLS-DA model of semen plantaginis, parched semen plantaginis and salted semen plantaginis, the independent variable accumulation interpretation ability parameter R 2 X is 0.985, and the dependent variable cumulative interpretation ability parameter R 2 Y is 0.938, predictive power parameter Q 2 And the values are 0.807 and are all larger than 0.5, which shows that the modeling type has strong interpretation rate and prediction rate, the obtained OPLS-DA score graph is shown in figure 7, and the Variable Importance Projection (VIP) value is shown in figure 8. As can be seen from FIG. 7, the crude product of semen plantaginis and the processed product show a clear phenomenon of classification and aggregation. As can be seen from FIG. 8, the VIP value>1 is a standard total extraction of 10 VIP values, and the importance ranks are sequentially peak 21 (acteoside), peak 24, peak 19 (plantain D), peak 26, peak 22 (rhoifolin), peak 8, peak 4 (geniposide), peak 27, peak 7, and peak 15, which may be the differential markers of semen plantaginis, parched semen plantaginis, and salted semen plantaginis.
2.4. Determination and analysis of chromaticity values of semen plantaginis and different processed products thereof
And (3) measuring the chromaticity value of the decoction piece powder of the semen plantaginis and different processed products by adopting a spectrocolorimeter. Taking 12 batches of semen plantaginis, stir-fried semen plantaginis and salted semen plantaginis decoction piece powder (passing through a third sieve) and uniformly spreading on a glass slide, pressing the powder to be about 1mm thick, adopting a D65 light source approved by the International Commission on illumination, taking white as a background, measuring the aperture to be 8mm, selecting 2 degrees from a visual angle, collecting decoction piece powder color images after the correction of the white board, measuring the chromaticity value for three times in parallel, and taking an average value, wherein the result is shown in Table 6.
L in Lab color mode * The value is a brightness index, a * 、b * Is a chromaticity index, wherein a * Is of the red-green axis b * Yellow Lan Zhou, the sample color was measured using the total chroma value (E * ) Expressed, the formula is E * ＝(L *2 +a *2 +b *2 ) 1/2 。ΔL * ＝L * -L 0 * 、Δa * ＝a * -a 0 * 、Δb * ＝b * -b 0 * Color difference delta E * ＝(ΔL *2 +Δa *2 +Δb *2 ) 1/2 The color change conditions of the raw materials to the processed products in brightness, red, green, yellow and blue and the overall color are expressed, and the calculated results are shown in Table 6. When delta E * At a value of 6 to 12, the color difference is visually recognized, and when delta E * >At 12, the color difference is significant. As can be seen from Table 7, the color difference values of the 12 batches of semen plantaginis and the two processed products are all>6.0, namely, the raw product and the processed product can be obviously distinguished in the same batch of medicinal materials, but the color difference value between the two processed products of the stir-fried plantain seed and the salted plantain seed is smaller, and the raw product and the processed product are difficult to distinguish by naked eyes. In addition, ΔL of semen plantaginis and parched semen plantaginis * At-14.12 to-2.52, average value of-9.54, and delta L of semen plantaginis and salinized semen plantaginis * -15.06 to-4.90, and the average value is-10.87, which indicates that the brightness of the processed plantain seeds is reduced and the color is deepened; Δa of semen plantaginis and parched semen plantaginis * 3.97-5.47, average value of 4.88, delta a of semen plantaginis and salinized semen plantaginis * 4.48 to 5.78, and the average value is 5.26, which indicates that the red color of the processed semen plantaginis is deepened; semen plantaginis, parched semen plantaginis, and salted semen plantaginis Δb * The positive and negative values are not large, which indicates that the color of the semen plantaginis on the yellow-blue axis is not changed greatly before and after processing.
TABLE 6 colorimetric value results
TABLE 7 color value variation results
2.5. In vitro antioxidant activity assay for semen plantaginis and different processed products thereof
2.5.1. Measurement method
Based on the pre-test, the concentration of the sample solution was diluted to 60. Mu.g/mL with 60% methanol, and the measurement was performed according to the method prescribed by the Grignard kit, and the procedure was as follows.
Preparation of a reagent solution:
(1) reagent one: the powder was dropped to the bottom by throwing several times before use, and each was dissolved by adding 0.49mL of distilled water.
(2) And (2) a reagent II: before use, the powder falls into the bottom by throwing a few times, and 2.86mL of distilled water is added into each powder to be fully dissolved for standby.
(3) Preparing an ABTS working solution: according to the sample size, the reagent I: the reagents II=1:1 are mixed in proportion, and after light-shielding reaction is carried out for 12 hours (the reaction is finished within two days), the mixture is diluted by 20 to 30 times by absolute ethyl alcohol for standby, and each reagent and the formula thereof are shown in table 8.
TABTS reaction System
Mixing the above solutions, standing in dark for 6min, transferring into a glass cuvette (optical path 1 cm), and reading absorbance at 734nm, respectively designated as A Blank space 、A Control And A Measurement And ABTS radical scavenging was calculated.
ABTS radical scavenging% = [1- (a) Measurement -A Control ）/A Blank space ]×100％。
2.5.2. Measurement results
The results of the antioxidant activity of the 12 batches of plantain seeds, the fried plantain seeds and the salted plantain seeds are shown in Table 9. As shown in Table 9, all of the 12 batches of semen plantaginis and its processed products have the ability to scavenge ABTS free radicals, wherein the fried semen plantaginis is the strongest, the difference between the salted semen plantaginis and the fried semen plantaginis is small, and the semen plantaginis raw products have the weakest scavenging ability.
TABLE 9 antioxidant Activity assay results
2.6. Analysis of spectrum-effect relationship of semen plantaginis and different processed products thereof
2.6.1. Partial Least Squares Regression (PLSR) analysis
The SIMCA14.1 software is used, the ABTS clearance of 12 batches of plantain seeds, stir-fried plantain seeds and salted plantain seeds is used as a dependent variable, the peak area of a common peak is converted and subtracted to be an independent variable (the peak area of a partial missing chromatographic peak is calculated as 0), and a partial least squares regression analysis (PLSR) is used for carrying out correlation analysis, so as to establish a regression equation.
The regression equation was established as y=0.059x 1 +0.095X 2 +0.001X 3 -0.016X 4 +0.048X 5 +0.058X 6 +0.030X 7 +0.090X 8 -0.036X 9 -0.007X 10 +0.075X 11 +0.066X 12 -0.095X 13 +0.008X 14 +0.061X 15 +0.084X 16 -0.008X 17 +0.047X 18 -0.112X 19 +0.007X 20 -0.051X 21 +0.005X 22 +0.013X 23 +0.080X 24 +0.131X 25 -0.060X 26 -0.018X 27 -0.164X 28 -0.066X 29 -0.052X 30 -0.055X 31 -0.092X 32 -0.114X 33 -0.114X 34 +40761, the normalized regression coefficients obtained are shown in FIG. 9. Wherein, the regression coefficients of the peaks 1-3, 5-8, 11, 12, 14-16, 18, 20 and 22-26 are larger than 0, which indicates that the regression coefficients of the peaks are positively correlated with the antioxidant activity, and the regression coefficients of the rest peaks are negatively correlated with the antioxidant activity.
2.6.2. Gray correlation (GRA) analysis
And (3) carrying out averaging treatment on data (the peak area of partial missing chromatographic peaks is calculated as 0) and antioxidant activity data after the peak areas of common peaks of the plantain seeds, the fried plantain seeds and the salted plantain seeds are reduced by water. The antioxidant activity data after treatment is set as a parent sequence x 0 (k) Peak areaData is subsequence x i (k) (k is peak number), and gray correlation coefficients of the parent sequence and the child sequence are calculated according to the formula (1). Wherein ρ is a resolution coefficient, the smaller ρ is, the larger the resolution is, and the value interval of ρ is [0,1]When ρ is less than or equal to 0.5463, the resolution is best, and ρ=0.5 is usually taken. The arithmetic average of the correlation coefficients is calculated according to the formula (2), namely the correlation degree, and the result is shown in the table 10.
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When the association degree is greater than 0.8, the association degree of the parent sequence and the child sequence is larger; when the association degree is between 0.6 and 0.8, the association degree is common; when the association degree is smaller than 0.6, the association degree of the two is smaller. As can be seen from Table 10, peaks 1 to 12, 15, 18 to 22, 24 to 26, 28, 29 and 34 have a large correlation with the contribution of antioxidant activity. By combining with PLSR results, peaks 1-3, 5-8, 11, 12, 15, 18, 20, 22 and 24-26 are positive correlation chromatographic peaks with high correlation degree of antioxidant activity of semen plantaginis and its processed products, namely components with main antioxidant effect, wherein peak 5 is protocatechuic acid, peak 18 is akebia phenylethanoid glycoside B, peak 22 is fisetin, and the other material components corresponding to the chromatographic peaks are yet to be further researched.
TABLE 10 correlation results
Comparative example 1
The fingerprints of psyllium, salted psyllium and fried psyllium were constructed in substantially the same manner as in example 1, except that the test solution was prepared as follows:
taking about 1.0g of each sample powder (passing through a second sieve), precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 60% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the weight loss with 60% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
The comparison diagram of the obtained corresponding characteristic map is shown in fig. 10. As can be seen from fig. 10, the peak (confirmed as geniposidic acid) with a retention time of about 3min, which is extracted with 60% methanol, has an insignificant signal of the remaining characteristic peaks due to an excessively large response value, and the number of characteristic peaks in the spectrum is significantly reduced, and compared with fig. 10, the information of the small and medium polar components in fig. 4 is more abundant, and at the same time, the influence of impurities is reduced by extraction and purification, so that the spectrum separation effect is better.
Comparative example 2
The fingerprint of psyllium was constructed in substantially the same manner as in example 1, except that the elution procedure (table 11) was adjusted, and the detection results corresponding to each elution procedure in table 11 are shown in fig. 11.
TABLE 11
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All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical scheme of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.
The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.
The above examples merely represent a few embodiments of the present application and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (12)
1. The method for constructing the fingerprint of the plantain seed, the salted plantain seed or the fried plantain seed is characterized by comprising the following steps:
providing a sample solution comprising the steps of: extracting the sample with an extraction solvent to obtain an extract; dispersing the extract in water, and adding ethyl acetate for extraction; the sample is semen plantaginis, salted semen plantaginis or parched semen plantaginis;
Taking the sample solution for liquid chromatography, wherein the conditions of the liquid chromatography comprise:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is employed, the procedure of which comprises:
0-5 min, wherein the volume percentage of the mobile phase A is increased from 10% to 16%,
5-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15-20 min, wherein the volume percentage of the mobile phase A is increased from 18% to 25%,
20-25 min, wherein the volume percentage of the mobile phase A is increased from 25% to 45%,
25-30 min, wherein the volume percentage of the mobile phase A is increased from 45% to 60%,
30-40 min, wherein the volume percentage of the mobile phase A is increased from 60% to 70%,
the volume percentage of the mobile phase A is reduced from 70% to 10% in 40-41 min,
41-50 min, wherein the volume percentage of the mobile phase A is maintained to be 10%.
2. The method for constructing a fingerprint according to claim 1, wherein the extraction solvent comprises methanol and the extraction method comprises ultrasound.
3. The method of claim 2, wherein the ultrasound conditions include at least one of:
(1) The power of the ultrasonic wave is 200W-300W,
(2) The frequency of the ultrasonic wave is 30 kHz-50 kHz, and
(3) The ultrasonic time is 50-70 min.
4. The method of claim 1, wherein the conditions for providing the test solution comprise at least one of:
(1) The volume ratio of the water to the ethyl acetate is 1: (1-3),
(2) The mass-volume ratio of the sample to the extraction solvent is 1 g/(20-30) mL, and
(3) The mass volume ratio of the test sample to the ethyl acetate is 1 g/(20-60) mL.
5. The method according to any one of claims 1 to 4, wherein in the liquid chromatography analysis:
0 min-10.5 min, the detection wavelength is 240 nm-250 nm,
10.5 min-35.5 min, the detection wavelength is 320 nm-340 nm,
35.5-50 min, and the detection wavelength is 240-250 nm.
6. The method according to any one of claims 1 to 4, wherein the conditions for liquid chromatography further comprise at least one of:
(1) The chromatographic column is a C18 chromatographic column,
(2) The chromatographic column has a length of 100 mm-150 mm, an inner diameter of 2 mm-2.2 mm and a particle size of 1.6 μm-1.9 μm,
(3) The flow rate is 0.28 mL/min-0.32 mL/min,
(4) Column temperature is 28-32 ℃, and
(5) The sample injection amount is 1-3 mu L.
7. The identification method of the plantain seed, the salted plantain seed and the stir-fried plantain seed is characterized by comprising the following steps:
taking the standard substances of the plantain seed, the salted plantain seed and the fried plantain seed as reference substances, and respectively preparing corresponding fingerprint patterns according to the construction method of any one of claims 1 to 6;
providing a solution to be tested, comprising the steps of: extracting the to-be-detected product with an extraction solvent to obtain an extract; dispersing the extract in water, and adding ethyl acetate for extraction;
performing liquid chromatography analysis on the solution to be detected to obtain a chromatographic spectrum of the to-be-detected product, and respectively comparing the chromatographic spectrum of the to-be-detected product with the fingerprint spectrum of the plantain seed reference product, the fingerprint spectrum of the salted plantain seed reference product and the fingerprint spectrum of the fried plantain seed reference product;
the conditions of the liquid chromatography include:
(1) Mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution with volume concentration of 0.05-0.2%, and
(2) Gradient elution is employed, the procedure of which comprises:
0 mm-5 min, the volume percentage of the mobile phase A is increased from 10% to 16%,
5 mm-15 min, the volume percentage of the mobile phase A is increased from 16% to 18%,
15 mm-20 min, the volume percentage of the mobile phase A is increased from 18% to 25%,
20 mm-25 min, the volume percentage of the mobile phase A is increased from 25% to 45%,
25 mm-30 min, the volume percentage of the mobile phase A is increased from 45% to 60%,
30 mm-40 min, the volume percentage of the mobile phase A is increased from 60% to 70%,
40 mm-41 min, the volume percentage of the mobile phase A is reduced from 70% to 10%,
41 mm-50 min, wherein the volume percentage of the mobile phase A is maintained to be 10%.
8. The method for constructing a fingerprint according to claim 7, wherein the extraction solvent comprises methanol and the extraction method comprises ultrasound.
9. The method of claim 8, wherein the ultrasound conditions include at least one of:
(1) The power of the ultrasonic wave is 200W-300W,
(2) The frequency of the ultrasonic wave is 30 kHz-50 kHz, and
(3) The ultrasonic time is 50-70 min.
10. The method of claim 7, wherein the conditions for providing the solution to be tested comprise at least one of:
(1) The volume ratio of the water to the ethyl acetate is 1: (1-3),
(2) The mass-volume ratio of the sample to the extraction solvent is 1 g/(20-30) mL, and
(3) The mass volume ratio of the test sample to the ethyl acetate is 1 g/(20-60) mL.
11. The method of construction according to any one of claims 7 to 10, wherein in the chromatographic analysis:
0 min-10.5 min, the detection wavelength is 240 nm-250 nm,
10.5 min-35.5 min, the detection wavelength is 320 nm-340 nm,
35.5-50 min, and the detection wavelength is 240-250 nm.
12. The method of construction according to any one of claims 7 to 10, wherein the chromatographic conditions further comprise at least one of:
(1) The chromatographic column is a C18 chromatographic column,
(2) The chromatographic column has a length of 100 mm-150 mm, an inner diameter of 2 mm-2.2 mm and a particle size of 1.6 μm-1.9 μm,
(3) The flow rate is 0.28 mL/min-0.32 mL/min,
(4) Column temperature is 28-32 ℃, and
(5) The sample injection amount is 1-3 mu L.
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| CN202310703454.4A CN116893227A (en) | 2023-06-14 | 2023-06-14 | Method for constructing fingerprints of semen plantaginis, salinized semen plantaginis and stir-fried semen plantaginis and identification method thereof |
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| CN202310703454.4A CN116893227A (en) | 2023-06-14 | 2023-06-14 | Method for constructing fingerprints of semen plantaginis, salinized semen plantaginis and stir-fried semen plantaginis and identification method thereof |
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Citations (4)
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|---|---|---|---|---|
| CN109444290A (en) * | 2018-12-21 | 2019-03-08 | 广东方制药有限公司 | The construction method and detection method of Asiatic plantain medicinal material UPLC characteristic spectrum |
| CN115541756A (en) * | 2022-10-08 | 2022-12-30 | 广东一方制药有限公司 | Fingerprint spectrum construction method, detection method and identification method of plantago Chinese medicinal material or preparation |
| CN115684382A (en) * | 2022-08-31 | 2023-02-03 | 广东一方制药有限公司 | Construction method and application of fingerprint of traditional Chinese medicinal materials of Plantago major, plantago major and mixed primordial plantain |
| CN115856108A (en) * | 2022-08-31 | 2023-03-28 | 广东一方制药有限公司 | Construction method and identification method of fingerprint of plantain herb traditional Chinese medicine formula granules |
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| CN109444290A (en) * | 2018-12-21 | 2019-03-08 | 广东方制药有限公司 | The construction method and detection method of Asiatic plantain medicinal material UPLC characteristic spectrum |
| CN115684382A (en) * | 2022-08-31 | 2023-02-03 | 广东一方制药有限公司 | Construction method and application of fingerprint of traditional Chinese medicinal materials of Plantago major, plantago major and mixed primordial plantain |
| CN115856108A (en) * | 2022-08-31 | 2023-03-28 | 广东一方制药有限公司 | Construction method and identification method of fingerprint of plantain herb traditional Chinese medicine formula granules |
| CN115541756A (en) * | 2022-10-08 | 2022-12-30 | 广东一方制药有限公司 | Fingerprint spectrum construction method, detection method and identification method of plantago Chinese medicinal material or preparation |
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